Pluripotent stem cells (PS), such as human pluripotent stem (hPS) cells, are capable of immortal proliferation in vitro and differentiation into derivatives of all three embryonic germ layers (Cohen D E, Melton D (2011) Nat Rev Genet 12: 243). As a result, the isolation of hPS cells, which include human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells (Blanpain et al. (2012) Nat Rev Mol Cell Biol 13: 471), has spurred new avenues of research to evaluate their potential to provide a renewable source of human cells for basic research and as replacement cells for the treatment of injury, aging, or any one of a number of intractable degenerative diseases such as osteoarthritis, cardiovascular disease, macular degeneration, Parkinson's and perhaps even Alzheimer's disease (Cohen D E, Melton D (2011) Nat Rev Genet 12: 243; Blanpain et al. (2012) Nat Rev Mol Cell Biol 13: 471). Reprogramming methods for creating iPS cells from somatic cells (Nakagawa (2010) Adv Exp Med Biol 695: 215) have greatly expanded the number and diversity of PS cell lines, including hPS cell lines, available for research. Donor-derived hPS cells are a source of patient matched cell types for disease modeling (Tiscornia et al. (2011) Nat Med 17: 1570), drug screening (Laposa (2011) J Cardiovasc Pharmacol 58: 240), and the development of potential autologous cell replacement therapies (Nelson et al. (2010) Stem Cells Cloning 3: 29). However, there remains a need for efficient directed differentiation methods and improved cell purification technologies for deriving various cell types with sufficient purity and known identity to meet the standards required for translation into routine clinical application.
Current directed differentiation methods for obtaining specific mature cell types from hPS cells are sometimes limited by low efficiencies of reproducibly yielding the desired cell types, and in certain instances, such preparations rarely exceed 30% purity (Cohen, Melton (2011) Nat Rev Genet 12: 243). One approach to increasing the yield is enrichment of desired cell types using one or more progenitor-specific markers. For example, cell enrichment using surface antigens that define progenitor populations has been used to improve the yield of the desired cell types such as neural and cardiomyocyte progenitors (Dubois et al. (2011) Nat Biotechnol 29: 1011; Yuan et al. (2011) PLoS One 6: e17540). Progenitor surface markers could also be useful for monitoring and validating hPS differentiation and for high throughput screening of reagents that stimulate differentiation toward a given lineage. However, apart from mapped hematopoietic progenitor markers, there is a paucity of validated cell surface antigens for most embryonic progenitor cell lineages.
Phage display is a powerful ligand selection method that has been applied both in vitro and in vivo for the identification of cell-specific targeting peptides (Molek et al. (2011) Molecules 16: 857; Teesalu et al. (2012) Methods Enzymol 503: 35). Peptide libraries displayed on phage particles are selected by repeated rounds of enrichment for target binding phage. Displayed peptides, genetically expressed on phage coat proteins, are identified by sequencing recovered phage DNA. A distinct advantage of phage display is that it is a non-biased approach that does not require prior knowledge of the targeted cell surface receptor. However, selection against a mixed population of differentiated hPS cells is challenging because the cellular heterogeneity limits the abundance of each of the various cell type specific surface targets. Clonal expansion of cells derived from hPS cell differentiation could provide a more abundant source of progenitor cell surface targets for phage selection. Over 140 distinct clonal embryonic progenitor cell lines have been derived from hES cells using a combinatorial cell cloning approach (the ACTCellerate Initiative) that resulted in a diverse assortment of clonally pure, scalable cell lines that were selected under a variety of cell culture and differentiation conditions (West et al. (2008) Regen Med 3: 287). Characterization of these clonal progenitors could result in the identification of markers associated with progenitor cell types of specific organs and tissues allowing for both enrichment of specific progenitors from a mixed population of cells, as well as the monitoring of the development potential of these progenitor cells both in vivo and in vitro.
The instant invention addresses a variety of needs known in the art, including, but not limited to, the identification of markers associated with progenitor cell lines and the enrichment of progenitor cell types for use in research and clinical applications.